Compositions and methods for enhancing cancer immunotherapy

ABSTRACT

A nutritional supplement containing fish oil and selenium has been identified that enhances immunotherapy of tumors, particularly in regard to reducing PDL-1 expression. A formulation for such a supplement that is both well tolerated and palatable is also provided.

This application is a continuation of U.S. patent application Ser. No.16/007,509, filed Jun. 13, 2018, which claims the benefit of U.S.Provisional Application No. 62/519,087 filed on Jun. 13, 2017, and ofU.S. patent application Ser. No. 16/007,539, filed Jun. 13, 2018, whichclaims the benefit of U.S. Provisional Application No. 62/519,093, filedon Jun. 13, 2017, U.S. Provisional Application No. 62/519,096, filed onJun. 13, 2017, U.S. Provisional Application No. 62/595,002 filed on Dec.5, 2017, and U.S. Provisional Application No. 62/670,275 filed on May11, 2018. These and all other referenced extrinsic materials areincorporated herein by reference in their entirety. Where a definitionor use of a term in a reference that is incorporated by reference isinconsistent or contrary to the definition of that term provided herein,the definition of that term provided herein is deemed to be controlling.

FIELD OF THE INVENTION

The field of the invention is cancer immunotherapy.

BACKGROUND

The background description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

Radiotherapy and chemotherapy protocols utilized in the treatment ofcancer can clearly benefit patients, but can be ineffective or lesseffective with some cancers. In addition both radiotherapy andchemotherapy are associated with significant side effects, includingnausea, weight loss, hair loss, damage to the gastrointestinal tract,and skin irritation.

Attempts have been made to enhance the effectiveness of radiotherapy.For example, gold nanoparticles that have been modified to target tumorcells have been used to enhance radiotherapy (Yang et al, ACS Nano,2014, 8(9):8992-9002). All publications herein are incorporated byreference to the same extent as if each individual publication or patentapplication were specifically and individually indicated to beincorporated by reference. Where a definition or use of a term in anincorporated reference is inconsistent or contrary to the definition ofthat term provided herein, the definition of that term provided hereinapplies and the definition of that term in the reference does not apply.Similarly, COX-2 inhibitors have been used to selectively sensitizetumor cells to the effects of radiation (Choy and Milas, J. Natl CancerInst (2003) 95(19):1140-1452). Such approaches, however, can have issueswith selectivity and may not be effective against all tumor types. Todate attempts to reduce the side effects of radiotherapy are primarilydirected to partitioning the total radiation dose into a number ofsmaller radiation doses (leaving time in between to allow for recovery),targeting of tumor using shielding, and identification of the boundariesof the tumor and localization of radiotherapy to that site.Unfortunately, such approaches can fail to adequately treat all of thetumor cells.

Attempts have also been made to enhance the effects of chemotherapy.Some studies have suggested that consumption of fish oil can improveresults from chemotherapy, however other research has suggested thatfish oil can interfere (Daenen et al, JAMA Oncol (2015) 1(3):350-358).Formulation of chemotherapeutic agents as nanoparticles has also beenattempted (Xu et al, Coll. Surf. B: Biointerfaces (2006) 48(1):50-57).It is unclear, however, if all chemotherapeutic drugs are suitable forsuch reformulation. Codelivery of chemotherapeutic drugs with siRNAdesigned to interfere with multi-drug resistance has also been explored.Such siRNAs, however, are sequence specific and may not be suitable forsome tumors.

Mitigation of the side effects of chemotherapy are generally directed atproviding symptomatic relief. For example, antiemetics can be used toreduce nausea, along with diet modification and eating small, frequentmeals that avoid certain foods. Unfortunately such approaches are notalways effective. In some instances chemotherapeutic agents are selectedto have reduced toxicity in order to reduce side effects, however suchagents may also have reduced effectiveness against tumor cells.

Thus, there is still a need for safe and effective compositions andmethods to enhance the effectiveness and/or reduce the side effects ofcancer radiotherapy and/or chemotherapy.

SUMMARY OF THE INVENTION

The inventive subject matter provides compositions and methods forenhancing and improving radiotherapy of tumors in a synergistic mannerthrough the use of a nutritional supplement containing fish oil andselenium.

One embodiment of the inventive concept is a method of treating a tumorby applying a radiotherapy protocol to a patient in need of treatmentwhile providing the patient with a nutritional supplement containingfish oil and selenium (for example, as shown in Table 1) in an amount toprovide a synergistic effect in reducing tumor volume or weight. In someembodiments the nutritional supplement is provided to the patient priorto the initiation of radiotherapy. Such a nutritional supplement can beformulated such that two or more of the components of the supplement areprovided in amounts as described in Table 1.

Another embodiment of the inventive concept is a method of reducing sidewhile providing the patient with a nutritional supplement formulatedthat includes fish oil and selenium (for example, as in Table 1) in anamount to reduce a side effect of the radiotherapy protocol. In someembodiments the nutritional supplement is provided to the patient priorto the initiation of radiotherapy. Such a nutritional supplement can beformulated such that two or more of the components of the supplement areprovided in amounts as described in Table 1.

Another embodiment of the inventive concept is a method of modulatinggene (e.g. an angiogenesis-related gene, an apoptosis-related gene,etc.) expression in a tumor, by providing a nutritional supplement to atumor or an animal having a tumor, wherein the nutritional supplementincludes fish oil and selenium (for example, as in Table 1), and wherethe nutritional supplement is provided in an amount to modulateexpression of a gene of the tumor. In some embodiments the nutritionalsupplement is provided to the patient prior to the initiation ofradiotherapy and during radiotherapy; in other embodiments such asupplement is provided during or at the initiation of the application ofradiotherapy. In preferred embodiments the nutritional supplement isformulated such that two or more components of the supplement areprovided in amounts as described in Table 1.

Another embodiment of the inventive concept is a method of reducingmetastasis from a tumor, by providing the patient having a metastatictumor with a nutritional supplement that includes fish oil and selenium(such as in Table 1) where the nutritional supplement is provided in anamount to reduce metastatic activity of the tumor. In some embodimentssuch a nutritional supplement is provided either prior to or at theinitiation of radiotherapy, and can be provided through the course ofradiotherapy. In a preferred embodiment the nutritional supplement isformulated such that two or more of the supplement's components areprovided in amounts as described in Table 1.

Another embodiment of the inventive concept is a method of reducingangiogenesis in a tumor, comprising providing the patient having a tumorwith a nutritional supplement that includes fish oil and selenium (forexample, as in Table 1) in combination with radiotherapy, where thenutritional supplement is provided in an amount to reduce an angiogenicactivity of the tumor. In some embodiments such a nutritional supplementis provided concurrent with the initiation of radiotherapy, and can beprovided to the patient during the application of radiotherapy. Inpreferred embodiments the nutritional supplement is formulated such thattwo or more of the supplement's components are provided in amounts asdescribed in Table 1.

Another embodiment of the inventive concept is a method of reducingincidences of cancer stem cells in a tumor, by providing the patienthaving a tumor with a nutritional supplement that includes fish oil andselenium (such as in Table 1), wherein the nutritional supplement isprovided in an amount to reduce the incidence of stem cells in thetumor. In a preferred embodiment the nutritional supplement is providedconcurrently with the application radiotherapy. The nutritionalsupplement can be formulated such that two or more components of thesupplement are provided in amounts as described in Table 1.

Various objects, features, aspects and advantages of the inventivesubject matter will become more apparent from the following detaileddescription of preferred embodiments, along with the accompanyingdrawing figures in which like numerals represent like components.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: FIG. 1 depicts a treatment protocol in which nutritionalsupplementation is provided starting either 7 days prior toimplementation of radiotherapy or simultaneously with tumor cellimplantation. Mice were sacrificed 21 days after tumor cellimplantation.

FIGS. 2A and 2B: FIG. 2A depicts changes in body weight over time forvarious treatment groups. FIG. 2B depicts change in body weight invarious treatment groups over the course of the study. Note that bodyweight is reported following removal of the primary tumor mass.

FIGS. 3A to 3D: FIGS. 3A to 3D show typical synergistic effects on tumorvolume and weight of co-treatment with radiotherapy and the supplementcontaining fish oil and selenium. FIG. 3A shows changes in tumor volumeover time for various treatment groups. FIG. 3B provides a detailed viewof the first 11 days of treatment as shown in FIG. 3A. FIG. 3C providesa histogram of tumor weight for various treatment groups. FIG. 3Dprovides photographs of exemplary tumors from various treatment groups.

FIGS. 4A to 4G: FIG. 4A: Effect of treatment using radiotherapy, asupplement containing fish oil and selenium, and combined treatment ongastrocnemius muscle mass 21 days after tumor cell injection. FIG. 4B:Effect of treatment using radiotherapy, a supplement containing fish oiland selenium, and combined treatment on soleus muscle mass 21 days aftertumor cell injection. FIG. 4C: Effect of treatment using radiotherapy,NutraWell supplement, and combined treatment on lung (inclusive ofmetastatic tumor) weight 21 days after tumor cell injection. FIG. 4D:Effect of treatment using radiotherapy, NutraWell supplement, andcombined treatment on liver (inclusive of metastatic tumor) weight 21days after tumor cell injection. FIG. 4E: Effect of treatment usingradiotherapy, NutraWell supplement, and combined treatment on spleen(inclusive of metastatic tumor) weight 21 days after tumor cellinjection. FIG. 4F provides quantitative results of studies of lungmetastasis in animal subjects treated with a supplement containing fishoil and selenium and/or radiotherapy. FIG. 4G shows the results ofstudies of Ki-67 expression in primary tumor sites and metastatic sitesin animal subjects treated with a supplement containing fish oil andselenium and/or radiotherapy.

FIGS. 5A to 5E: FIG. 5A: Effect of treatment using radiotherapy,NutraWell supplement, and combined treatment on platelet count 21 daysafter tumor cell injection. FIG. 5B: Effect of treatment usingradiotherapy, NutraWell supplement, and combined treatment on whiteblood cell count 21 days after tumor cell injection. FIG. 5C: Effect oftreatment using radiotherapy, NutraWell supplement, and combinedtreatment on lymphocyte count 21 days after tumor cell injection. FIG.5D: Effect of treatment using radiotherapy, NutraWell supplement, andcombined treatment on granulocyte count 21 days after tumor cellinjection. FIG. 5E: Effect of treatment using radiotherapy, NutraWellsupplement, and combined treatment on neutrophil/lymphocyte percentageratio 21 days after tumor cell injection.

FIGS. 6A and 6B: FIG. 6A: Effect of treatment using radiotherapy,NutraWell supplement, and combined treatment on serum albumin 21 daysafter tumor cell injection. FIG. 6B: Effect of treatment usingradiotherapy, NutraWell supplement, and combined treatment on serumcreatinine 21 days after tumor cell injection.

FIGS. 7A and 7B: FIG. 7A: Effect of treatment using radiotherapy,NutraWell supplement, and combined treatment on serum IL-6 21 days aftertumor cell injection. FIG. 7B: Effect of treatment using radiotherapy,NutraWell supplement, and combined treatment on serum IL-1β 21 daysafter tumor cell injection.

FIGS. 8A to 8G: FIG. 8A: Effect of treatment using radiotherapy,NutraWell supplement, and combined treatment on expression of the VEGFgene in an implanted tumor 21 days after tumor cell injection. FIG. 8B:Effect of treatment using radiotherapy, NutraWell supplement, andcombined treatment on expression of the BAX gene in an implanted tumor21 days after tumor cell injection. FIG. 8C: Effect of treatment usingradiotherapy, NutraWell supplement, and combined treatment on expressionof the Bcl-2 gene in an implanted tumor 21 days after tumor cellinjection. FIG. 8D: Effect of treatment using radiotherapy, NutraWellsupplement, and combined treatment on expression of the caspase 3 genein an implanted tumor 21 days after tumor cell injection. FIG. 8E:Effect of treatment using radiotherapy, NutraWell supplement, andcombined treatment on expression of the BAX gene in lung 21 days aftertumor cell injection. FIG. 8F: Effect of treatment using radiotherapy,NutraWell supplement, and combined treatment on expression of the Bcl-2gene in lung 21 days after tumor cell injection.

FIG. 8G: Effect of treatment using radiotherapy, NutraWell supplement,and combined treatment on expression of the caspase 3 gene in lung 21days after tumor cell injection.

FIGS. 9A and 9B: FIG. 9A depicts a testing protocol in which micereceive radiotherapy on days 8, 10, and 12, with nutritionalsupplementation beginning with the initiation of radiotherapy. Mice aresacrificed at day 14 or at day 24 following implantation of tumor cells.FIG. 9B: Treatment groups derived from the protocol shown in FIG. 9A.

FIGS. 10A to 10C: FIG. 10A: Serum albumin concentration at 14 days and24 days following tumor cell implantation in mice treated as shown inFIG. 9A. FIG. 10B: Lymphocyte counts at 14 days and 24 days followingtumor cell implantation in mice treated as shown in FIG. 9A. FIG. 10C:N/L Ratio at 14 days and 24 days following tumor cell implantation inmice treated as shown in FIG. 9A.

FIG. 11A to 11D: FIG. 11A: VEGF expression within the tumor mass 24 daysfollowing tumor cell implantation in mice treated as shown in FIG. 9A.FITC represents VEGF-specific staining. FIG. 11B: VEGF expression withinthe lung (metastasis) 24 days following tumor cell implantation in micetreated as shown in FIG. 9A. FITC represents VEGF-specific staining.FIG. 11C: EGFR expression within the lung (metastasis) 24 days followingtumor cell implantation in mice treated as shown in FIG. 9A. FITCrepresents EGFR-specific staining. FIG. 11D: EGFR expression within thetumor mass 24 days following tumor cell implantation in mice treated asshown in FIG. 9A. FITC represents EGFR-specific staining.

FIG. 12: CD31 (cancer stem cell marker) expression within the tumor massand in lung tissue (metastasis) 24 days following tumor cellimplantation in mice treated as shown in FIG. 9A. FITC representsCD31-specific staining.

FIGS. 13A and 13B: FIG. 13A shows the presence of H1F1-a (hypoxiamarker) protein within the tumor mass and in lung tissue (metastasis) 24days following tumor cell implantation in mice treated as shown in FIG.9A. FITC represents H1F1-α-specific staining. FIG. 13B shows results ofgene expression studies of tumor samples from subjects treated with anutritional supplement containing fish oil and selenium and/orradiotherapy.

FIG. 14: Expression of apoptosis markers 24 days following tumor cellimplantation in mice treated as shown in FIG. 9A.

FIGS. 15A and 15B: FIG. 15A shows the results of studies characterizingPDL-1 gene expression in primary and metastatic tumor sites in animalmodels of human disease treated with a supplement containing fish oiland selenium and/or radiotherapy. FIG. 15B shows the results of studiescharacterizing PD-1 gene expression in primary and metastatic tumorsites in animal models of human disease treated with a supplementcontaining fish oil and selenium and/or radiotherapy.

FIGS. 16A and 16B: FIG. 16A depicts a testing protocol in which micereceive radiotherapy on days 8, 10, and 12, with nutritionalsupplementation provided prior to tumor cell implantation, at the timeof tumor cell implantation, or at the initiation of radiotherapy. Miceare sacrificed at day 21 following implantation of tumor cells. FIG.16B: Treatment groups derived from the protocol shown in FIG. 16A.

FIG. 17: Photomicrographs of cross sections of the intestines of micetreated by the protocol described in FIG. 16A.

FIGS. 18A to 18F: FIG. 18A: Effect of treatment using radiotherapy,NutraWell supplement, and combined treatment on expression of the VEGFgene 21 days after tumor cell injection. FIG. 18B: Effect of treatmentusing radiotherapy, NutraWell supplement, and combined treatment onexpression of the BAX gene 21 days after tumor cell injection. FIG. 18C:Effect of treatment using radiotherapy, NutraWell supplement, andcombined treatment on expression of the Bcl-2 gene 21 days after tumorcell injection. FIG. 18D: Effect of treatment using radiotherapy,NutraWell supplement, and combined treatment on expression of theCaspase 3 gene 21 days after tumor cell injection. FIG. 18E: Effect oftreatment using radiotherapy, NutraWell supplement, and combinedtreatment on expression of the Bcl-2 gene 21 days in lung (i.e.metastasis) after tumor cell injection. FIG. 18F: Effect of treatmentusing radiotherapy, NutraWell supplement, and combined treatment onexpression of the Caspase 3 gene 21 days in lung (i.e. metastasis) aftertumor cell injection.

FIGS. 19A and 19B: FIG. 19A depicts a testing protocol in which micereceive radiotherapy on days 8, 10, and 12, with nutritionalsupplementation provided for 7 days prior to tumor cell implantation.Mice are sacrificed at day 24 following implantation of tumor cells.FIG. 19B: Treatment groups derived from the protocol shown in FIG. 19A.

FIGS. 20A and 20B: FIG. 20A: Effect of a nutritional supplementcontaining fish oil and selenium on body mass in mice receiving repeatedradiotherapy following tumor cell implantation using the protocol shownin FIG. 19A. FIG. 20B: Effect of treatment using radiotherapy, anutritional supplement containing fish oil and selenium, and combinedtreatment on gastrocnemius muscle mass 21 days after tumor cellinjection using the protocol shown in FIG. 19A.

FIGS. 21A and 21B: FIG. 21A depicts a testing protocol mice in whichmice receive radiotherapy on days 8, 10, and 12, with nutritionalsupplementation provided for 7 days prior to, the day of, or 8 daysafter tumor cell implantation. Mice are sacrificed at day 21 followingimplantation of tumor cells. FIG. 21B: Treatment groups derived from theprotocol shown in FIG. 21A.

FIG. 22: Effect of NutraWell supplementation on body mass in micereceiving repeated radiotherapy following tumor cell implantation usingthe protocol shown in FIG. 21A.

FIG. 23: Changes in tumor volume over time on repeated radiotherapy incombination with NutraWell supplement in mice treated as in the protocolshown in FIG. 21A.

FIGS. 24A and 24B: FIG. 24A: Effect of treatment using repeatedradiotherapy in combination with NutraWell supplement, and combinedtreatment on serum TNF-α 21 days after tumor cell injection in micetreated using the protocol shown in FIG. 21A. FIG. 24B: Effect oftreatment using repeated radiotherapy in combination with NutraWellsupplement, and combined treatment on serum IL-6 21 days after tumorcell injection in mice treated using the protocol shown in FIG. 21A.

DETAILED DESCRIPTION

The following description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

The inventive subject matter provides compositions and methods in whicha nutritional supplement that includes fish oil and selenium (forexample, a supplement containing fish oil, selenium derived fromselenium yeast, and certain vitamins, minerals, amino acids, andsaccharides, e.g. “NutraWell”) is used in combination with radiotherapy.Combination therapy with radiation and such a supplement surprisinglyprovides a significant synergistic effect in reduction of tumor size. Inaddition, side effects of radiotherapy (e.g. neutropenia, loss of bodymass, loss of muscle mass, damage to acyl cells of the gastrointestinaltract, etc.) are reduced and/or mitigated relative to application ofradiotherapy without use of such a supplement. Surprisingly, expressionof genes related to angiogenesis and apoptosis were also found to bemodulated in tumor cells on use of a supplement containing fish oil andselenium, both with and without the application of radiotherapy. Inaddition, metastasis is prevented and the growth and spread of cancerstem cells was found to be reduced.

One should appreciate that the disclosed techniques provide manyadvantageous technical effects including enhancing the effectiveness ofcurrent radiotherapeutic protocols used in the treatment of cancer whilereducing the side effects associated with these approaches.

The following discussion provides many example embodiments of theinventive subject matter. Although each embodiment represents a singlecombination of inventive elements, the inventive subject matter isconsidered to include all possible combinations of the disclosedelements. Thus if one embodiment comprises elements A, B, and C, and asecond embodiment comprises elements B and D, then the inventive subjectmatter is also considered to include other remaining combinations of A,B, C, or D, even if not explicitly disclosed.

In some embodiments, the numbers expressing quantities of ingredients,properties such as concentration, reaction conditions, and so forth,used to describe and claim certain embodiments of the invention are tobe understood as being modified in some instances by the term “about.”Accordingly, in some embodiments, the numerical parameters set forth inthe written description and attached claims are approximations that canvary depending upon the desired properties sought to be obtained by aparticular embodiment. In some embodiments, the numerical parametersshould be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and parameters setting forth the broad scopeof some embodiments of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspracticable. The numerical values presented in some embodiments of theinvention may contain certain errors necessarily resulting from thestandard deviation found in their respective testing measurements.

In one embodiment of the inventive concept, a nutritional supplementcomposition as shown in Table 1 (“Nutrawell”) is provided to improve theresults of radiotherapy.

TABLE 1 Component Minimum Maximum Unit Maltodextrin 10000 50000 mg WheyProtein Isolate 5000 60000 mg Whey Protein Concentrate 1000 50000 mgFructooligosaccharides/Inulin 40 15000 mg Granulated Honey 1000 9000 mgOat Fiber 500 15000 mg Natural French Vanilla Flavor 500 20000 mg SoyProtein 500 50000 mg Brownulated Powdered Brown Sugar 500 10000 mgNatural Vanilla Masking Flavor 500 5000 mg Lecithin 200 10000 mg Milk,Non-fat 50 5000 mg Rice Protein Powder 50 5000 mg Calcium Caseinate 502000 mg Oils Flax Seed Oil 100 7000 mg Canola Oil 100 7000 mg Borage Oil100 7000 mg Olive Oil 100 7000 mg Fish Oil 150 10,000 mg Pure Lemon Oil100 1000 mg Pure Orange Oil 50 1000 mg Mixed Tocopherols 0.5 200 mgVitamins/Minerals Potassium Phosphate 200 1500 mg Calcium Carbonate 1005000 mg Choline Bitartrate 150 2500 mg Sodium Chloride 100 2000 mgCalcium Phosphate Tribasic 100 2000 mg Ascorbic Acid 50 3000 mgPotassium Chloride 50 2000 mg Magnesium Oxide 50 500 mg Selenium Yeast30 4000 mcg Chromium Yeast 30 3000 mcg Molybdenum Yeast 30 2000 mcgInositol 10 5000 mg Zinc Sulfate Monohydrate 5 200 mg Dry Vitamin EAcetate 5 2000 IU Niacinamide 5 500 mg Ferric Orthophosphate 3 100 mgCalcium Pantothenate 3 200 mg Manganese Sulfate Monohydrate 3 100 mgBeta Carotene 1 100 mg Copper Gluconate 1 15 mg Vitamin D3 25 5000 IUVitamin K2 2 1000 mcg Pyridoxine HCl 0.5 200 mg Potassium Iodide 0.51500 mg Riboflavin 0.5 1000 mg Thiamine Hydrochloride 0.5 2500 mg DryVitamin K1 1 500 mcg Vitamin A Acetate 500 100000 IU Folic Acid 10010000 mcg d-Biotin 10 10000 mcg Vitamin B12 1 3000 mcg Amino AcidsL-Carnitine 300 30000 mg L-Glutamine 500 60000 mg L-Arginine Base 50030000 mg Taurine 50 2000 mg L-Lysine 50 2000 mg Alpha Lipoic Acid 101000 mg Resveratrol 15 1500 mg Co-Enzyme Q10 10 5000 mg Glycine 5 1000mg Proline 5 1000 mg Bacterial Cultures Lact. Acidophilus (app. 10billion total) 2 500 mg Bifido Bifidium (app. 10 billion total) 2 500 mgLac. Bulgaricus (app. 10 billion total) 2 500 mg Bifido Longum (app. 10billion total) 2 500 mg Strep. Thermophilus (app. 10 billion total) 2500 mg Enzymes Papain 5 100 mg Pepsin 5 100 mg Lipase 5 100 mg Bromelain5 100 mg Pancreatin 4X 0.5 100 mg Lactase 1 100 mg Betaine HCl 3 100 mgPlant Products Pineapple Juice Powder 2 500 mg Papaya Fruit Powder 2 500mg Quercetin 30 3000 mg EGCG 25 600 mg OPC 15 500 mg Anthocyanins 155000 mg Ellagic Acid 10 300 mg Astaxanthin 2 90 mg Fucoidan 20 1500 mgMushroom Preparation Cordyceps 5 6000 mg Ganoderma Lucidum 15 10000 mgShiitake 40 15000 mg Maitake 30 15000 mg Turkey Tail 30 15000 mg

The composition shown in Table 1 includes components that have variousphysiological and biochemical effects, including anti-inflammatoryactivity, lowering of blood glucose levels, lowering of cholesterol, andanti-tumor activity. Other components provide supplementation ofnecessary vitamins, minerals, and amino acids at elevated levels. Othercomponents (e.g. enzymes, lecithin) serve to aid in digestion andabsorption of components of the composition when consumed. Thecombination of these complementary activities provides a synergisticeffect that exceeds the simple additive effect of individual components.It should be appreciated that the composition shown in Table 1 alsoincludes certain flavorants (e.g. brown sugar, honey, vanilla flavor andmasking agent) that serve to improve palatability and acceptance.Certain components (e.g. honey, brown sugar, milk, rice protein, casein)can provide both flavor and caloric energy. The Inventor has found thatthe combination of flavorants described above is effective in providingcompliance with consumption of the nutritional supplement in effectiveamounts. In some embodiments, such flavorants can be excluded withoutnegatively impacting the effectiveness of the nutritional supplement.

Components shown in Table 1 can be provided as a single formulation (forexample, as a pill, tablet, capsule, powder, liquid, suspension, etc.)or can be segregated into different formulations (for example, as pills,tablets, capsules, powders, liquids, suspensions, or combinationsthereof). The amounts shown in Table 1 are exemplary, and representtypical daily dosages provided to an adult of normal stature andotherwise normal health. These amounts can be adjusted to account fordifferences in body mass, gender, medical condition, etc. For example, arelatively small patient weighing 40 kilos or less may receive benefitfrom dosages provided at or below the low end of the ranges provided,whereas a relatively large patient weighing 100 kilograms or more mayrequire dosages provided at the high end of the ranges noted (or more).In some embodiments such a daily dose can be distributed as multipledoses throughout the day. In some of such embodiments the composition ofeach of such distributed doses can be identical. In other embodimentsthe composition of such distributed doses can be different, provided thesummation of such doses provides the required supplementation.

In an exemplary embodiment human tumor cells (following transplantationinto nude mice) are treated with 1 gram per day of the nutritionalsupplement, radiotherapy, or 1 gram per day of the nutritionalsupplement and chemotherapy. The mice are weighed during treatment tocharacterize side effects such as nausea and loss of appetite. Afterseveral weeks the mice are sacrificed and the tumor characterized. Tumorvolume is determined, and the impact of therapy on organ and musclevolume is determined. The degree of neutropenia is also characterized. Atypical protocol is shown in Table 2.

TABLE 2 Sacrifice (21st day) 1 C Control N = 6 2 T Tumor N = 6 3 PTNTumor + Nutrawell (−7 day start) N = 6 4 TN Tumor + Nutrawell (0 daystart) N = 6 5 TR Tumor + Radiotherapy (3 Gy × 3) N = 6 6 PTRN Tumor +Radiotherapy (3 Gy × 3) + N = 6 Nutrawell (−7 day start) 7 TRN Tumor +Radiotherapy (3 Gy × 3) + N = 6 Nutrawell (8 day start)

Body Weight/Wasting

A typical treatment schedule is depicted schematically in FIG. 1. Somesubjects received treatment with a supplement containing fish oil andselenium (PTN, PTRN) prior to tumor cell implantation, with a portion ofthese (PTRN) receiving radiotherapy. Other subject started treatmentwith such a supplement at the time of tumor cell implantation (TN, TRN),with some of these receiving radiotherapy (TRN). Results of body weightstudies compared to control subjects (C) and subjects implanted withtumor cells and otherwise untreated (T) are shown in FIGS. 2A and 2B. Asshown, mice receiving both radiotherapy and the supplement containingfish oil and selenium gained weight at a significantly greater rate thanthose receiving only radiotherapy, indicating a reduction in sideeffects normally associated with this treatment mode. Pre-treatment witha nutritional supplement containing fish oil and selenium had aparticularly pronounced effect.

Tumor Size

FIGS. 3A to 3D show typical synergistic effects on tumor volume andweight of co-treatment with radiotherapy and a supplement containingfish oil and selenium for similar treatment groups in a murine model ofhuman cancer. FIG. 3A shows the effect of various treatment protocols ontumor volume over the course of 3 weeks, with FIG. 3B providing a scaledview of the effect over the initial 11 days. As shown, treatment with asupplement containing fish oil and selenium alone provides anapproximately 60% reduction in tumor volume. Treatment with radiationalone provides a similar reduction in tumor volume. In the absence of asynergistic effect one would therefore anticipate a reduction in tumorvolume to approximately 25% of that of the untreated tumor.Surprisingly, what is observed is a greater than 90% reduction in tumorvolume to approximately 7% of the untreated tumor-indicative of asignificant synergistic effect. FIG. 3C shows that similar effects arefound when tumor weight is characterized. FIG. 3D shows photographstypical examples of tumors excised from test animals followingtreatment; the effects of cotherapy with a nutritional supplementcontaining fish oil and selenium with radiotherapy is readily apparenton visual inspection. As such, it is apparent that cotherapy with asupplement containing fish oil and selenium and radiotherapy can providea synergistic effect in reducing tumor volume and/or mass.

Muscle Wasting

Side effects of radiotherapy go beyond loss of appetite and weight loss,and can include damage to internal organs, loss of muscle mass, anemia,neutropenia, reduction in kidney function, etc. To determine theprotective effects of cotherapy with a supplement containing fish oiland selenium in regards to such side effects on muscle mass and organweight of treated mice were also characterized following therapy. Theresults are shown in FIGS. 4A to 4E. FIGS. 4A and 4B show the effects oftreatment with a supplement containing fish oil and selenium,radiotherapy, and cotherapy with radiotherapy using such a supplement(with and without supplement pre-treatment) on the weight ofgastrocnemius muscle and soleus muscle (respectively), with test groupsdesignated as described above. As shown, cotherapy with such asupplement and radiotherapy provides almost complete retention of muscleweight.

Metastasis

FIGS. 4C, 4D, and 4E show the effects of treatment as described above onthe weight lungs inclusive of any metastatic tumors (FIG. 4C), liversinclusive of any metastatic tumors (FIG. 4D), and spleens inclusive ofany metastatic tumors (FIG. 4E). As shown lack of treatment leads to anincrease in weight, due at least in part to the presence of metastatictumors. This increase is not entirely mitigated by radiotherapy alone(TR), however subjects receiving treatment with a supplement containingfish oil and selenium had organ weights similar to those of controlsubjects, both with and without cotherapy with radiotherapy.

Similarly, quantitation of metastatic sites within the lungs of animalmodels of human disease treated with a nutritional supplement containingfish oil and selenium and/or radiotherapy. Typical metastatic sites areshown in the photographs in the left panel of FIG. 4F, and enumerated ina histogram in the right panel. As shown, treatment with either such asupplement (TN) or radiotherapy (TR) reduced the number of metastaticsites to some extent, however treatment with both the supplement andradiotherapy (TRN) resulted in a complete lack of apparent metastaticsites in most subjects.

Without wishing to be bound by theory, the Inventor believes that thiseffect on metastasis (and also on tumor size and mass, as shown above)can be due to a synergistic effect provided by the use of a nutritionalsupplement containing fish oil and selenium in combination withradiotherapy on tumor cell proliferation. As shown in FIG. 4G, theexpression of the proliferation marker Ki-67 is suppressed in theprimary tumor site on treatment with either a supplement containing fishoil and selenium (TN), on treatment with radiotherapy (TR), or treatmentusing both modes (TRN). Metastatic sites (right panel) show a relatedtrend, with treatment with the nutritional supplement or radiotherapyalone resulting in a moderate reduction in the expression of thisproliferation marker, but surprisingly showing a synergistic effect inreducing expression of Ki-67 in metastatic sites. This indicates thatuse of a supplement containing fish oil and selenium can reduce tumormetastasis, including metastasis that is not prevented by radiotherapy,and can do so in a synergistic manner.

Neutropenia/Anemia

Anemia and neutropenia are often found in cancer and as side effects ofradiotherapy, due at least in part to suppression of bone marrowactivity. The effects of cotherapy with a supplement containing fish oiland selenium on various blood cell populations are shown in FIGS. 5A to5E. FIG. 5B shows the effects of a nutritional supplement containingfish oil and selenium, radiotherapy, and cotherapy with such asupplement and radiotherapy on white blood cells as shown in FIG. 5B,and lymphocytes as shown in FIG. 5C. This indicates that a nutritionalsupplement containing fish oil and selenium can be useful to improve redblood cell, granulocyte, white blood cell, and/or lymphocyte suppressionresulting from the presence of a tumor and from radiotherapy utilized inthe treatment of such tumors.

As shown in FIG. 5A, platelets were found to be elevated in untreatedanimals bearing tumors (T). Platelet concentrations were reduced bytreatment with either a supplement containing fish oil and selenium orradiotherapy, but were most pronounced in animals receiving cotherapywith such a supplement and radiotherapy. This effect was particularlypronounced in subjects receiving pre-treatment with a supplementcontaining fish oil and selenium, which reduced platelet concentrationsto the values found for control animals. Granulocyte concentrations weresimilarly elevated in untreated tumor bearing animals (as shown in FIG.5D), and were further elevated in such animals receiving onlyradiotherapy. Cotherapy with a nutritional supplement containing fishoil and selenium and radiotherapy, was found to reduce this effect. Asshown in FIG. 5E, tumor bearing animals show an elevatedneutrophil/lymphocyte ratio (NLR) relative to control animals, which iseven more elevated in similar subjects receiving only radiotherapy.Treatment with a supplement containing fish oil and selenium was foundto be effective in shifting this ratio towards a more normal value, bothas a monotherapy and as a part of cotherapy with radiotherapy. Thisindicates that a nutritional supplement containing fish oil and seleniumcan be useful in reducing platelet concentration, granulocyteconcentration, and/or neutrophil/lymphocyte ratios that are elevated dueto the presence of a tumor and due to radiotherapy used to treat such atumor.

Serum Proteins

The effects of cotherapy with a supplement containing fish oil andselenium on various serum biochemistry markers, which provideinformation regarding kidney function, liver function, and/ornutritional status are shown in FIGS. 6A and 6B. FIG. 6A shows theeffects of a nutritional supplement containing fish oil and selenium,radiotherapy, and cotherapy with such a supplement and radiotherapy onan animal tumor model. As shown, serum albumin concentration (anindicator of nutritional status) is reduced in untreated tumor-bearinganimals, and is only slightly improved by radiotherapy alone. Treatmentwith a nutritional supplement containing fish oil and selenium, eitheras a monotherapy or as cotherapy with radiotherapy, improves serumalbumin concentration—particularly when such a supplement is provided asa pretreatment. This is improved by cotherapy with a nutritionalsupplement containing fish oil and selenium. This indicates thattreatment with a nutritional supplement containing fish oil andselenium, either as a monotherapy or as cotherapy with radiotherapy, canimprove the nutritional status of tumor-bearing subjects.

FIG. 6B shows the results of similar studies where serum creatinine (ameasure of kidney function) is characterized. As shown, untreated tumorbearing subjects shown elevated creatinine concentrations indicative ofkidney damage. This is marginally improved by radiotherapy alone.Cotherapy with a nutritional supplement containing fish oil and seleniumand radiotherapy, however, shows a synergistic effect in reducing serumcreatinine concentrations—particularly when such a nutritionalsupplement is provided as a pretreatment.

Cytokines

The growth and spread of tumors is associated with inflammation, as isthe application of radiotherapy. Surprisingly, Inventors have found thatcotherapy with a supplement containing fish oil and selenium iseffective in reducing the concentration of pro-inflammatory cytokines,indicating that such co-treatment is effective in reducing inflammationassociated with tumors, and with radiotherapy of tumors. The effects ofNutraWell supplement on serum concentrations of pro-inflammatorycytokines is shown in FIGS. 7A and 7B. FIG. 7A shows the concentrationof IL-6 in control animals, untreated tumor-bearing animals, and tumorbearing animals treated with a nutritional supplement containing fishoil and selenium, radiotherapy, or such a supplement and radiotherapy incombination. As shown, untreated tumor-bearing animals show a largeincrease in serum IL-6 concentration. This is only somewhat reduced byradiotherapy. Use of a nutritional supplement containing fish oil andselenium, either as a monotherapy or as cotherapy with radiotherapy, wasfound to reduce serum IL-6 concentrations in tumor-bearing animals. FIG.7B shows the results of similar studies of IL-1β. The results with IL-1(3 are similar to those found for IL-6. This indicates that anutritional supplement containing fish oil and selenium can effectivelyreduce serum concentrations of inflammation-associated cytokines intumor-bearing subjects, either as a monotherapy or as cotherapy withradiotherapy. The Inventor believes that such reduction is accompaniedby a reduction in inflammation in such animals.

Tumor Gene Expression

Surprisingly, Inventors have also found that treatment with anutritional supplement containing fish oil and selenium can modify geneexpression in tumor cells in vivo, and can provide a synergistic effectto such changes in gene expression resulting from radiotherapy. In someembodiments the genes are related to cytokines and/or are related toapoptosis. Examples of the effect of radiotherapy, treatment with anutritional supplement containing fish oil and selenium, and cotherapywith radiotherapy on gene expression in implanted tumor cells in vivoare shown in FIG. 8A to 8G. FIG. 8A shows the results of such treatmentson the expression of VEGF in tumor cells. As shown, monotherapy with anutritional supplement containing fish oil and selenium and radiotherapyreduced VEGF expression. Cotherapy with a nutritional supplementcontaining fish oil and selenium and radiotherapy provided dramaticallyreduced expression of VEGF.

FIGS. 8B and 8D show the effects of treatment with a nutritionalsupplement containing fish oil and selenium, radiotherapy, and cotherapywith such a supplement and radiotherapy on tumor BAX expression andmetastatic (lung) tumor BAX expression, respectively. BAX is considereda marker for apoptosis. As shown, BAX expression in untreated tumor islow, and is not impacted by radiotherapy. Monotherapy with a nutritionalsupplement containing fish oil and selenium resulted in dramaticincreases in BAX expression, and elevated BAX expression when used ascotherapy with radiotherapy (particularly with the supplement wasprovided as a pretreatment). As shown in FIGS. 8C and 8E, Bcl-2expression was found to be elevated in tumor cells and metastatic (lung)tumor cells (respectively), and was reduced by either treatment with anutritional supplement containing fish oil and selenium or radiotherapyas monotherapies. Greater reductions in Bcl-2 expression were found whensuch a supplement and radiotherapy were used as cotherapies. Expressionof caspase 3 (which is associated with apoptosis) is elevated in tumors(FIG. 8D) treated with radiotherapy or with a nutritional supplementcontaining fish oil and selenium alone, and is also elevated bycotherapy with such a supplement as radiotherapy (particularly when thesupplement is provided as a pre-treatment). As shown in FIG. 8F,expression of caspase 3 is reduced in metastatic (lung) tumors relativeto lung tissue of control subjects. As shown, caspase 3 expression insuch tumors is increased by radiotherapy or treatment with a nutritionalsupplement containing fish oil and selenium, as well as cotherapy.

Repeated Radiotherapy

Similar studies were performed using modified treatment protocols. Onemodified treatment protocol utilizing multiple rounds of radiotherapy,as is typical with human radiotherapy, is shown in FIGS. 9A and 9B.

Results from serum albumin and blood cell characterization studiesfollowing treatment with NutraWell, radiotherapy, and combined treatmentwith a nutritional supplement containing fish oil and selenium andradiotherapy using the protocol shown in FIG. 9A are shown in FIGS. 10Ato 10C. As shown in FIG. 10A serum albumin concentration is reduced intumor bearing animals, particularly at later time points. This isimproved by treatment with a nutritional supplement containing fish oiland selenium or radiotherapy as monotherapies, and by cotherapy. Asshown in FIG. 10B, tumor bearing animals showed suppressed lymphocytecounts relative to control subjects, particularly at later time points.This was only marginally improved by radiotherapy alone, howevertreatment with a nutritional supplement containing fish oil and selenium(either as a monotherapy or in combination with radiotherapy) waseffective in increasing lymphocyte concentration—particularly in latertime points. FIG. 10C shows the results of similar studies wherein theneutrophil/lymphocyte ratio (NLR) was characterized. As shown, animalsbearing tumors show a dramatic elevation in this value at later timepoints. This is reduced by treatment with a nutritional supplementcontaining fish oil and selenium, radiotherapy, and cotherapy with sucha supplement and radiotherapy.

Surprisingly, treatment with a nutritional supplement containing fishoil and selenium, radiotherapy, and combined treatment with such asupplement and radiotherapy using the protocol shown in FIG. 9A also hasan impact on both expression of tumor cell markers and tumor cellmetastasis. In the following studies the tumor cells selected forimplantation are derived from a lung tumor and have a strong tendency tometastasize to the lung from the implantation site. FIGS. 11A to 11Dshow the results of immunocytochemistry studies of different tissuesfrom mice treated with the protocol shown in FIG. 9A. It is notable thatNutraWell supplementation alone reduces or eliminates metastasis.

As shown in FIG. 11A, VEGF expression within the tumor of untreated mice(left panel, FITC staining) is evident. Treatment with either anutritional supplement containing fish oil and selenium or radiotherapyalone dramatically reduced VEGF expression, as does cotherapy. Numericalresults are provided in the right panel of FIG. 11A. Similar results arefound in tumors that have metastasized to the lungs, as shown in FIG.11B. As shown, VEGF expression is apparent in untreated metastatictumors (left panel, FITC staining) and is sharply reduced in animalsthat received treatment with a nutritional supplement containing fishoil and selenium, radiotherapy, or both. Numerical results of thesestudies are provided in the right panel of FIG. 11B.

Similar results are found with the expression of EGFR, overexpression ofwhich is associated with tumors. As shown in FIG. 11C, elevatedexpression of EGFR (left panel, FITC staining) is evident in untreatedtumors, and is reduced in subjects treated with a nutritional supplementcontaining fish oil and selenium, radiotherapy, or cotherapy with such asupplement and radiotherapy. Numerical results are shown in the rightpanel of FIG. 11C. Similar results are found for EGFR expression oftumors that have metastasized to the lungs, as shown in FIG. 11D. Asshown, elevated EGFR expression (left panel, FITC staining) is evidentin untreated metastatic sites, and is reduced in subjects treated with anutritional supplement containing fish oil and selenium or radiotherapyas monotherapies, and with cotherapy using such a supplement andradiotherapy. This suggests that use of a nutritional supplementcontaining fish oil and selenium, radiotherapy, and/or a combination oftreatment with such a supplement and radiotherapy can be effective inenhancing EGFR-directed treatment protocols in individuals receivingtreatment for cancer.

Surprisingly, treatment with a nutritional supplement containing fishoil and selenium, radiotherapy, and combined treatment with such asupplement and radiotherapy using the protocol shown in FIG. 9A also hasan impact on tumor stem cells. Such stem cells are associated withmetastasis and the development of resistance to various cancertherapies. In the following studies the tumor cells selected forimplantation are derived from a lung tumor and have a strong tendency tometastasize to the lung from the implantation site. FIG. 12 shows theresults of immunocytochemistry studies of different tissues from micetreated with the protocol shown in FIG. 9A. The left panel of FIG. 12shows the results of staining for CD31 (FITC staining), a stem cellmarker, in tumor cells. The right panel shows similar results formetastatic cells in the lungs. Untreated subjects show numerous cellswith elevated expression of CD31. Surprisingly, treatment with anutritional supplement containing fish oil and selenium in the absenceof radiotherapy reduces or eliminates the occurrence of cancer stemcells both in the tumor implantation site and at the lung metastaticsite.

Treatment with a nutritional supplement containing fish oil andselenium, radiotherapy, and combined NutraWell supplementation andradiotherapy using the protocol shown in FIG. 9A also has an impact onhypoxia often found in or among tumor cells. HIF1-α is a markerassociated with hypoxia. In the following studies the tumor cellsselected for implantation are derived from a lung tumor and have astrong tendency to metastasize to the lung from the implantation site.FIG. 13A shows the results of immunocytochemistry studies of differenttissues from mice treated with the protocol shown in FIG. 9A. The leftpanel shows the results of immunocytochemistry staining for HIF1-α (FITCstaining) in tumor cells, while the right panel shows the results ofsimilar staining at lung metastatic sites. Untreated subjects shownumerous cells with elevated levels of HIF1-a. Surprisingly, treatmentwith a nutritional supplement containing fish oil and selenium in theabsence of radiotherapy reduces or eliminates the occurrence of suchhypoxia markers both in the tumor implantation site and at the lungmetastatic site. FIG. 13B shows typical numerical results from a similarstudy where gene expression in tumor samples was characterized. Asshown, both treatment with a supplement containing fish oil and selenium(TN) and pre-treatment with a nutritional supplement containing fish oiland selenium (PTN) for one week reduce tumor expression of HIF1-a, asdoes radiotherapy (TR). When radiotherapy and such a nutritionalsupplement are used in combination (PTRN, TRN) a dramatic reduction intumor HIF1-α expression is evident, particularly in pretreated subjects(PTRN) (which show a synergistic effect). It's evident that use of anutritional supplement containing fish oil and selenium in combinationwith radiotherapy can reduce both HIF1-α protein content and geneexpression in tumor sites, potentially rendering them more vulnerable tohypoxic conditions.

Treatment with a nutritional supplement containing fish oil andselenium, radiotherapy, and combined supplementation and radiotherapyusing the protocol shown in FIG. 9A also has an impact on apopticactivity in tumor cells. Results of qPCR studies for expression ofvarious apoptosis markers (Bax, Bcl-2, and caspase 3) at 24 days fromtumor cell implantation in mice is shown in FIG. 14. As shown, theBax/Bcl-2 expression ratio is low in tumor cells, and only marginallyimproved by radiotherapy. Surprisingly, treatment with a nutritionalsupplement containing fish oil and selenium alone provided a significantincrease in this ratio. In addition, a pronounced synergistic effect wasfound on cotherapy with such a supplement and radiotherapy. Caspase 3expression was actually somewhat suppressed by radiotherapy alone, butwas dramatically increased by treatment with a nutritional supplementcontaining fish oil and selenium, both as a monotherapy and as cotherapywith radiotherapy.

Treatment with a nutritional supplement containing fish oil and seleniumin combination with radiotherapy has also been found to modulateexpression of PDL-1 and PD-1 in primary tumor sites and metastatic(lung) tumor sites in animal models of human disease. As shown in FIG.15A, both primary (left panel) and metastatic (right panel) tumor cellsshow high levels of PDL-1 expression (T). Surprisingly, these aremarkedly reduced by treatment with a nutritional supplement containingfish oil and selenium, both when pretreated (PTN) and treated at thetime of implantation (TN). As such, the Inventor believes that suchsupplements can be used to enhance immunotherapeutic approaches tocancer treatment. Treatment with radiotherapy (TR) also shows areduction in PD-L1 gene expression in both primary and metastatic sites,suggesting that radiotherapy alone can be used to enhanceimmunotherapeutic approaches to cancer treatment. Cotherapy with such anutritional supplement and radiotherapy is effective in reducing PDL-1expression in both primary tumor sites and metastatic sites, whether thesupplement is provided prior to implantation (PTRN) or at the time ofimplantation (TRN). Surprisingly, the effects are more marked at theprimary tumor site than at metastatic sites. This suggests thatcotherapy with a nutritional supplement that includes fish oil andselenium in combination with radiotherapy can render tumor cells at bothprimary and metastatic sites more susceptible to the patient's immunesystem, and/or can enhance the effect of immunotherapy approaches tocancer treatment.

Similar studies were performed on PD-1 expression. As shown in FIG. 15B,PD-1 expression is reduced in primary site tumor cells treated withradiotherapy (TR) and enhanced in primary site tumor cells (left panel)in animals treated with a supplement containing fish oil and selenium,either prior to implantation (PTN) or upon implantation (TN). Cotherapywith the nutritional supplement and radiotherapy provides differentialresults depending on if the supplement is provided prior to implantation(PTRN) or at the time of implantation (TRN). As shown in the right panelof FIG. 15B, tumor cells at metastatic sites showed reduced PD-1expression relative to samples taken from control animals. Surprisingly,radiotherapy resulted in an increase in PD-1 expression at metastaticsites (right panel), as did treatment with a supplement containing fishoil and selenium either prior to implantation or at the time ofimplantation. Cotherapy with radiotherapy and such a supplement providedhigher levels of PD-1 expression than those observed with monotherapy.It is apparent that treatment with a nutritional supplement containingfish oil and selenium, particularly in combination with radiotherapy,can shift PD-1 expression away from reduced expression levels seen intumor-bearing subjects, particularly in metastatic sites.

Preimplantation, Preradiotherapy, and Initiation of RadiotherapySupplementation with Repeated Radiotherapy

Another treatment protocol combining both pretreatment with anutritional supplement containing fish oil and selenium and multiplerounds of radiotherapy typical of clinical application is shown in FIGS.16A and 16B. In this protocol treatment with the supplement wasinitiated prior to tumor cell implantation, at the time of tumor cellimplantation, and at the initiation of radiotherapy.

Loss of intestinal absorption and the resulting malnutrition are a wellknown side effect of radiotherapy, particularly repeated radiotherapy.FIG. 17 shows photomicrographs that demonstrate the effect treatmentwith a nutritional supplement containing fish oil and selenium on thecellular architecture of the gut during radiotherapy. Cellulararchitecture of the gut is shown for untreated control subjects (topleft panel), tumor implanted subjects treated with multiple rounds ofradiotherapy (to right panel), tumor implanted subjects pretreated witha nutritional supplement containing fish oil and selenium andsubsequently treated with radiotherapy (lower left panel), and tumorimplanted subjects treated with a nutritional supplement containing fishoil and selenium at the time of initiation of radiotherapy (lower rightpanel). As shown, treatment with a nutritional supplement containingfish oil and selenium can maintain the intestinal brush border duringradiotherapy, with pretreatment apparently enhancing the intestinalbrush border. This indicates that treatment with such a supplement,particularly pretreatment, can effectively address side effects ofradiotherapy.

Such a treatment protocol has been found to modify expression of certaingenes in tumor cells in vivo. Results of qPCR studies of gene expression(e.g. angiogenic factor-related, apoptosis-related, etc.) in tumor cellsfrom mice treated by the protocol shown in FIG. 16A are shown in FIGS.18A to 18F. As shown in FIG. 18A, treatment with a nutritionalsupplement containing fish oil and selenium reduces VEGF expression intumor cells, as does repeated radiotherapy. Combined therapy with such asupplement and radiotherapy provides an enhanced reduction in VEGFexpression, particularly when a nutritional supplement containing fishoil and selenium is provided prior to initiation of radiotherapy.

FIG. 18B shows that BAX expression in tumor cells is increased bytreatment with a nutritional supplement containing fish oil andselenium, and relatively unaffected by repeated radiotherapy alone.Pretreatment with such a supplement in combination with repeatedradiotherapy also increased BAX expression. Similar results are foundfor expression of another apoptosis-related gene (Caspase 3), as shownin FIG. 18D. Results of studies of Caspase-3 expression in metastatic(lung) tumors are shown in FIG. 18F. As shown, expression of Caspase-3in such metastatic tumors is elevated by treatment with a nutritionalsupplement containing fish oil and selenium and by repeatedradiotherapy, and is similar to that of control cells when thesupplement is provided as cotherapy prior to initiation of radiotherapy.

Expression of another apoptosis-related gene, Bcl-2, in tumor cells isreduced by treatment with a nutritional supplement containing fish oiland selenium and by repeated radiotherapy alone (see FIG. 18C). Thisreduction in Bcl-2 expression is more pronounced on cotherapy with sucha supplement and repeated radiotherapy, particularly when the supplementis provided prior to initiation of radiotherapy. FIG. 18E shows theresults of similar studies performed on metastatic (lung) tumors. Asshown, the reduction in Bcl-2 expression is reduced in a synergisticfashion on cotherapy with a nutritional supplement containing fish oiland selenium and repeated radiotherapy, particularly when the supplementis provided prior to initiation of radiotherapy.

Pre-Implantation Supplementation and Repeated Radiotherapy

FIGS. 19A and 19B show a treatment protocol and related test groups(respectively) where a nutritional supplement containing fish oil andselenium is provided 7 days prior to implantation of tumor cells, withradiotherapy taking place on days 8, 10, and 12 following implantation.Mice were sacrificed on day 24 following implantation.

A well known side effect of both cancer and repeated radiotherapy isweight loss. This can be due to wasting associated with disease and withside effects of radiotherapy. The effect of treatment with a nutritionalsupplement containing fish oil and selenium on loss of body mass and onmuscle mass following repeated radiotherapy is shown in FIGS. 20A and20B. It should be appreciated that body mass was characterized followingremoval of the tumor mass. As shown in FIG. 20A, when tumor mass isremoved a significant decrease in remaining body mass is apparentrelative to control subjects. This is improved by treatment with anutritional supplement containing fish oil and selenium and byradiotherapy as monotherapies. Surprisingly, gains in body mass surpassthose of control subjects when such a supplement and repeatedradiotherapy are used in combination. FIG. 20B shows the weight ofgastrocnemius muscle in the various test groups. Loss of muscle mass isapparent in untreated tumor bearing animals. This is marginally improvedby repeated radiotherapy alone. Surprisingly, treatment with anutritional supplement containing fish oil and selenium providessignificant retention in muscle mass. Cotherapy with a nutritionalsupplement containing fish oil and selenium and repeated radiotherapyprovides a synergistic improvement in muscle mass relative to untreatedtumor bearing subjects. It is apparent that pre-treatment with anutritional supplement containing fish oil and selenium effectivelyreverses the loss of body mass (relative to control subjects) and musclemass resulting from both the presence of the tumor and repeatedradiotherapy.

Preimplantation Supplementation and Repeated Radiotherapy

Another treatment protocol with related treatment groups is shown inFIGS. 21A and 21B, which is similar to the protocol shown in FIGS. 16Aand 16B. In this protocol radiotherapy is provided on days 8, 10, and 12following implantation of tumor cells. Treatment with a nutritionalsupplement containing fish oil and selenium is provided either 7 daysprior to implantation, the day of implantation, or on the initiation ofradiotherapy. Mice were sacrificed on day 21 following tumor cellimplantation.

A well known side effect of both cancer and radiotherapy is weight loss.The effect of treatment with a nutritional supplement containing fishoil and selenium on loss of body mass and loss of muscle mass whenprovided prior to and when provided coincident with the initiation ofrepeated radiotherapy is shown in FIG. 22. It should be appreciated thatbody mass was characterized following removal of the tumor mass. Asshown, body weight gains over time of untreated tumor bearing subjects(following excision of the tumor) is dramatically reduced relative tocontrol subjects. Similar losses are noted on repeated radiotherapy whenused alone. Surprisingly, treatment with a nutritional supplementcontaining fish oil and selenium provides remarkably improved bodyweight gains over time, both as a monotherapy and when used incombination with repeated radiotherapy. This is particularly evidentwith such a supplement is used as a pretreatment.

Inventors have also found that pre-treatment with a nutritionalsupplement containing fish oil and selenium enhances the reduction intumor volume seen on repeated radiotherapy, as shown in FIG. 23. Asshown, both repeated radiotherapy and treatment with a nutritionalsupplement containing fish oil and selenium have a moderate effect inreducing tumor volume relative to tumors of untreated tumor bearinganimals. Surprisingly, tumor volume shows almost no change over timewhen repeated radiotherapy and a nutritional supplement containing fishoil and selenium are used as cotherapies, indicating a synergisticeffect.

The presence of tumors and repeated radiotherapy can also result in thedevelopment of inflammation, which can be characterized by the presenceof pro-inflammatory cytokines in serum. FIGS. 24A and 24B show theeffect of administration of a nutritional supplement containing fish oiland selenium with repeated radiotherapy on the concentration ofpro-inflammatory cytokines in mice treated as in the protocol shown inFIG. 21A. FIG. 24A shows values for serum TNF-α. It is apparent thatuntreated tumor bearing animals show highly elevated concentrations ofTNF-α, which is reduced to some extent by repeated radiotherapy.Treatment with a nutritional supplement containing fish oil and seleniumalso resulted in a reduction in serum TNF-α, particularly when such asupplement was used in cotherapy with repeated radiotherapy. FIG. 24Bshows the results of similar studies where the serum concentration ofIL-6 was characterized, which show similar results.

It should be apparent to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the inventive concepts herein. The inventive subjectmatter, therefore, is not to be restricted except in the spirit of theappended claims. Moreover, in interpreting both the specification andthe claims, all terms should be interpreted in the broadest possiblemanner consistent with the context. In particular, the terms “comprises”and “comprising” should be interpreted as referring to elements,components, or steps in a non-exclusive manner, indicating that thereferenced elements, components, or steps may be present, or utilized,or combined with other elements, components, or steps that are notexpressly referenced. Where the specification claims refers to at leastone of something selected from the group consisting of A, B, C . . . andN, the text should be interpreted as requiring only one element from thegroup, not A plus N, or B plus N, etc.

What is claimed is:
 1. A method of enhancing anti-tumor immunotherapy inan individual, comprising: applying an anti-tumor immunotherapy protocolto the patient; and providing the patient with a nutritional supplementcomprising fish oil and selenium.
 2. The method of claim 1, wherein thenutritional supplement is provided to the patient prior to initiation ofthe anti-tumor immunotherapy protocol.
 3. The method of claim 1, whereinthe nutritional supplement is provided to the patient concurrently withthe anti-tumor immunotherapy protocol.
 4. The method of claim 1, whereinthe nutritional supplement is formulated such that a plurality ofcomponents of the supplement are provided in amounts as described inTable
 1. 5. The method of claim 1, wherein the nutritional supplementcomprises three or more components as provided in Table
 1. 6. The methodof claim 1, wherein the nutritional supplement is provided in an amountsufficient to reduce PDL-1 expression in a cancer cell.